Solid-phase chemistry: Its principles and applications in clinical analysis.

A review is given of the development of solid-phase reaction systems (test papers, impregnated-fibre systems, multi-layer film systems) for rapid field and laboratory testing in clinical analysis.

Immobilized glutamate oxaloacetate transaminase. Steady state kinetic analysis and stability studies.

1. Glutamate oxaloacetate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) was immobilized on amino ethyl cellulose using the bifunctional reagent diethyl adipimidate. 2. The steady state kinetic analysis was performed for the particulate and the free enzyme, and the Michaelis constants measured for the amino ethyl cellulose derivative were not greatly different from those measured for the free glutamate oxaloacetate transaminase, while the latter were in good agreement with values in the literature. 3. The amino ethyl cellulose-glutamate oxaloacetate transaminase was slightly more stable than the free enzyme at 65 degrees C, but was stabilised less by polyethylene glycol than the free enzyme.

The preparation of nylon-tube-supported hexokinase and glucose 6-phosphate dehydrogenase and the use of the co-immobilized enzymes in the automated determination of glucose.

Triethyloxonium tetrafluoroborate was used to O-alkylate nylon-tube thus producing the imidate salt of the nylon which was further made to react with 1,6-diaminohexane. 2. Hexokinase (EC 2.7.1.1) and glucose 6-phosphate dehydrogenase (EC 1.1.1.49) were immobilized on the amino-substituted nylon tube through glutaraldeyde and bisimidates. 3. The effect of varying the conditions of O-alkylation and the amount of enzyme immobilized on the activity of nylon tube-hexokinase derivatives was determined. 4. The effect of varying the amount of enzyme immobilized on the activity of nylon-tube-glucose 6-phosphate dehydrogenase derivatives was determined. 5. The thermal stability of nylon-tube-hexokinase and nylon-tube-glucose 6-phosphate dehydrogenase derivatives was studied. 6. Different ratios of hexokinase and glucose 6-phosphate dehydrogenase were co-immobilized on nylon tube, and the rate of conversion of glucose into 6-phosphogluconolactone was compared with the individual activities of the immobilized enzymes. 7. Hexokinase and glucose 6-phosphate dehydrogenase co-immobilized on nylon tube were used in the automated analysis of glucose.

The preparation of several new nylon tube-glucose oxidase derivatives and their incorporation into the "reagentless" automated analysis of glucose.

Nylon tube was activated by alkylation with dimethyl sulfate and used for the immobilization of glucose oxidase. Lysine, hexamethylene diamine and polyethylene imine were also attached to activated nylon tube, and these nylon tube-spacer derivatives were reactivated with either glutaraldehyde or ethyl adipimidate for the subsequent coupling of glucose oxidase. The activities of all of the different nylon tube-glucose oxidase derivatives were compared by their incorporation into standard Technicon automated analysis systems. Activities were measured either spectrophotometrically, by following the production of hydrogen peroxide using an acid/KI assay, or polarographically by following the decrease in the oxygen concentration using a flow-through oxygen electrode assembly. The activity and stability of all of the nylon tube-glucose oxidase derivatives was such that their use in the routine estimation of glucose levels was an attractive proposition.

Preparation of some immobilized linked enzyme systems and their use in the automated determination of disaccharides.

1. Glucose oxidase (EC 1.1.3.4), amyloglucosidase (EC 3.2.1.3), invertase (EC 3.2.1.26) and beta-galactosidase (EC 3.2.1.23) were covalently attached via glutaraldehyde to the inside surface of nylon tube. 2. The linked enzyme system, comprising invertase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of sucrose. 3. The linked enzyme system, comprising beta-galactosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of lactose. 4. The linked enzyme system, comprising amyloglucosidase immobilized within a nylon tube acting in series with glucose oxidase immobilized in a similar way, was used for the automated determination of maltose. 5. Mixtures of glucose oxidase and amyloglucosidase were immobilized within the same piece of nylon tube and used for the automated determination of maltose. 6. Mixtures of glucose oxidase and invertase were immobilized within the same piece of nylon tube and used for the automated determination of sucrose.

The immobilization of enzymes on nylon structures and their use in automated analysis.

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.

The preparation and properties of yeast beta-fructofuranosidase chemically attached to polystyrene.

1. A process is described for the chemical attachment of an enzyme to the surface of a polystyrene matrix. 2. By this process yeast beta-fructofuranosidase was chemically attached to the surface of both polystyrene beads and polystyrene tubes. 3. The kinetics of sucrose hydrolysis by beta-fructofuranosidase and polystyrene-supported beta-fructofuranosidase were compared. 4. The pH-activity curve of the polystyrene-supported enzyme shows a marked difference from that of the free enzyme in solution. 5. The inhibitor dissociation constant with respect to tris is increased, whereas the inhibitor dissociation constant with respect to aniline is decreased when the enzyme is attached to polystyrene. 6. Differences between the properties of the bound and free enzyme are discussed in terms of a micro-environmental effect arising from the hydrophobic nature of the polystyrene support.

Some changes in the reactivity of enzymes resulting from their chemical attachment to water-insoluble derivatives of cellulose.

1. Purified ficin was chemically attached to CM-cellulose, and partially purified ATP-creatine phosphotransferase was chemically attached to both CM-cellulose and p-aminobenzylcellulose. 2. The apparent K(m) with respect to ATP and Mg(2+) of ATP-creatine phosphotransferase was observed to increase about tenfold on attachment of the enzyme to CM-cellulose, and to increase by only 23% on its attachment to p-aminobenzylcellulose. 3. The reactivity of both ficin and ATP-creatine phosphotransferase with 5,5'-dithiobis-(2-nitrobenzoic acid) was observed to decrease on chemical attachment of these enzymes to water-insoluble derivatives of cellulose. With derivatives prepared from CM-cellulose, the extent of the reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) was dependent on ionic strength, but with similar derivatives prepared from p-aminobenzylcellulose the extent of this reaction was independent of ionic strength. 4. The effect of diffusion and electrostatic interaction of charged enzyme substrates and charged enzyme supports on the apparent K(m) of a water-insoluble derivative of an enzyme is discussed. An equation is derived that satisfactorily describes the observed effects of these factors on the apparent K(m).

The kinetics of carboxymethylcellulose--ficin in packed beds.

1. The kinetics of the hydrolysis of benzoylarginine ethyl ester in packed columns of CM-cellulose-70-ficin and CM-cellulose-90-ficin were studied. 2. The apparent Michaelis constant, K'(m), of these preparations was calculated and shown to be dependent on the flow rate at low rates of perfusion through the columns. 3. The values for k(3) of these preparations were calculated and shown to be nearly independent of flow rate. 4. A modified form of the integrated Michaelis rate equation was used to describe the action of these materials and its limitations are discussed. 5. The hydrolysis of solutions of casein by these columns was studied.

The preparation and properties of ficin chemically attached to carboxymethylcellulose.

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.